DNA site-specific recombinases (SSRs) are a powerful tool for analyzing gene function in eukaryotes. SSRs recognize specific DNA sequences (recognition sites) and catalyze recombination between two recognition sites. Upon binding to their recognition sites, SSRs can induce conditional gene inactivation or expression. If the two recognition sites are located on the same DNA molecule in the same orientation, the intervening DNA sequence is excised by the SSR from the parental molecule as a closed circle, leaving one recognition site on each of the reaction products. If the two sites are in inverted orientation, the recognition-site flanked region is inverted through recombinase-mediated recombination. Alternatively, if the two recognition sites are located on different molecules, recombinase-mediated recombination will lead to integration of a circular molecule or translocation between two linear molecules. These features make SSRs extremely useful for a number of applications in eukaryotic systems, including conditional activation of transgenes, chromosome engineering to obtain deletions, translocations or inversions, removal of selection marker genes, gene replacement, targeted insertion of transgenes, and the activation or inactivation of genes by inversion (see e.g., Branda, et al., Dev. Cell. 6 (1):7-28 (2004); Nagy, Genesis. 26 (2):99-109 (2000); Cohen-Tannoudji et al., Mol. Hum. Reprod. 4(10):929-938 (1998)). The simultaneous use of multiple SSRs allows for the analysis of multiple gene knockouts or conditional gene expression.
The first widely used SSR in mammalian cultured cells and animals was the P1 bacteriophage-derived Cre gene (Sauer et al., Proc. Natl. Acad. Sci. U.S.A. 85 (14):5166-70 (1988); O'Gorman et al., Science. 251 (4999):1351-5 (1991); Lakso et al., Proc. Natl. Acad Sci. U.S.A. 89 (14):6232-6 (1992); Orban et al., Proc. Natl. Acad. Sci. U.S.A. 89 (15):6861-5 (1992)). Cre recognizes homotypic 34 base pair (bp) DNA sequences known as loxP sites and can induce the deletion, insertion, or inversion of DNA sequences depending on the number and orientation of loxP sites (Hoess et al., Proc. Natl. Acad. Sci. U.S.A. 9 (11):3398-402 (1982)). In addition to Cre, other SSRs have been shown to exhibit some activity in mammalian cells. These include the Kw recombinase of Kluyveromyces waltii (Ringrose et al., Eur. J. Biochem. 15:248 (3):903-12 (1997)); mutant integrases of phage lamda (Lorbach et al., J. Mol. Biol. 296 (5):1175-81 (2000)); the integrases of phage HK022 (Kolot et al., Mol. Biol. Rep. 26 (3):207-13 (1999)); mutant gammadelta resolvase (Schwikardi et al., FEBS. Lett. 471 (2-3):147-50 (2000)); beta-recombinase (Diaz et al., J. Biol. Chem. 274 (10):6634-40 (1999)); and ΦC31 from Streptomyces lividans (Groth et al., Proc. Natl. Acad. Sci. USA. 97 (11):5995-6000 (2000); Belteki et al., Nat. Biotechnol. 21 (3):321-4 (2003)).
FLP from Saccharomyces cerevisiae is another SSR that has been used in mammals (Dymecki, Proc. Natl. Acad. Sci. U.S.A. 93 (12):6191-6 (1996)). Similar to Cre, FLP recognizes a distinct 34 bp sequence known as an FRT site, and can mediate the deletion, inversion, and insertion of DNA sequences between two of these sites (McLeod et al., Mol. Cell Bio. 6 (10):3357-67 (1986)). Initial use of FLP in mouse and mammalian cells revealed inefficient recombinase activity due to thermo-instability of the protein (Buchholz et al., Nucleic Acids Res. 24 (21):4256-62 (1996)). Subsequent screening for thermo-stable mutants resulted in the identification of an enhanced FLP recombinase (FLPe), which showed a 4-fold increase in recombination efficiency compared to endogenous FLP (Buchholz et al., Nat. Biotechnol. 16 (7):657-62 (1998)). Despite this improvement in thermo-stability, the recombination efficiency of FLP in mammalian cultured cells remains quite low. FLP has only been shown to exhibit at most a 6% recombination rate in mouse embryonic stem (ES) cell clones, with mosaic recombination found in almost all ES clones (Schaft et al., Genesis. 31 (1):6-10 (2001)). This low efficiency of recombination has hampered the use of FLP in cultured cells.
One reason for the low efficiency of these SSRs in mammalian cells may be their prokaryotic origin. For use in eukaryotic systems, SSRs should ideally be expressed at high levels. Often, achieving high steady-state expression levels of prokaryotic genes in mammalian systems can be difficult. One potential problem is that the amino acid codon usage differs greatly between prokaryotes and vertebrates (Ikemura, Mol. Biol. Evol. 2 (1):13-34 (1985)). Prokaryotic genes often contain a proportionally high-abundance of codons for tRNAs that are rare in vertebrates, resulting in low levels of expression (Grantham et al., Nucleic Acids Res. 9 (1):r43-r74 (1981)). A second potential problem associated with expression of prokaryotic genes in vertebrates is the presence of cryptic splice acceptor/donor sites, since prokaryotic genes do not normally undergo splicing in the native host. Another potential problem is that a high number of the DNA dinucleotide motif CpG may also result in gene silencing, since DNA methylation occurs at such cytosines in vertebrates. Additionally, the overall base composition of the prokaryotic gene can affect mRNA stability in eukaryotic cells. Prokaryotic genes with high A/T content often result in less stable mRNAs and thus low levels of expression.
Optimization of the endogenous gene can be used to improve expression, however, codon-optimization has to be performed individually for each new gene, taking into account all factors that can influence gene expression. Codon-optimized Cre genes with improved expression in mammals have been described previously (e.g., Koresawa et al., Transplant Proc. 32 (7):2516-17 (2000); PCT International Publication No. WO/2002/04609). A codon-optimized ΦC31 recombinase has also been reported. (U.S. Patent Publication No. 20030186291).
The present invention overcomes previous shortcomings in the art by providing a codon-optimized FLP (FLPo) SSR and methods for its use. This genetically engineered FLP gene displays a marked increase in recombination efficiency compared to the native FLP gene and is therefore useful in a wide array of molecular applications.